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siRNA and mutant cloning vector prepared based on RNAi technology and rescue principle

A technology of cloning vectors and RNA interference, which is applied in the biological field and can solve problems such as immature key technologies

Active Publication Date: 2014-01-15
SHANGHAI GENECHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are not many reports on the application of RNA interference recovery. The main reason is that the key technology for obtaining mutant overexpression vectors is not mature enough.

Method used

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  • siRNA and mutant cloning vector prepared based on RNAi technology and rescue principle
  • siRNA and mutant cloning vector prepared based on RNAi technology and rescue principle
  • siRNA and mutant cloning vector prepared based on RNAi technology and rescue principle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of mutant cloning vector

[0039] 1. Preparation principle

[0040] The target gene used in this example is the SPP1 (NM_000582.2) gene. Prepare a double-stranded DNA oligo sequence expressing the interference sequence with restriction sites at both ends, and directly connect it into the interference vector; then transfer the ligated product to Enter the prepared bacterial competent cells, and perform PCR identification on the grown clones. After sequencing and comparison, the positive clones are the successfully constructed target gene RNA interference lentiviral vectors.

[0041] The mutated lentiviral overexpression vector carries out a synonymous mutation at the third base in the triplet codon of all coding amino acids in the RNAi target sequence of the target gene, that is, although the base sequence of the target gene changes, due to the A synonymous mutation occurs in the RNAi target mRNA region of the mRNA, so the amino acid sequence expre...

Embodiment 2

[0192] Verification of embodiment 2 RT-PCR method to mutant cloning vector

[0193] 1. Cell Transfection

[0194] 1.1 Cell plating: Trypsinize the 293T cells in the logarithmic growth phase and spread them into 24-well plates to ensure that the cell density reaches 80% on the second day before they can be used for transfection.

[0195] 1.2 Plasmid transfection: set negative control group (Lv-Scr-siRNA plasmid transfection group, obtained in Example 1), RNAi plasmid transfection group (SPP1-shRNA-plasmid), RNAi plasmid (SPP1-shRNA-plasmid) and RNAi rescue plasmid (SPP1-res-plasmid) co-transfection group, the next day, 1ug ​​negative control plasmid, 1ug ​​RNAi plasmid, 1ug ​​RNAi plasmid and 1ug RNAi rescue plasmid were transfected into 293T cells, the transfection reagent was Lipo2000 from Invitrogen, For the transfection method, refer to the Lipo2000 instructions.

[0196] 1.3 Identification of transfection efficiency: On the fourth day, the cell transfection efficiency wa...

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Abstract

The invention relates to the technical field of biology, and specifically relates to siRNA and a mutant cloning vector prepared based on an RNAi technology and a rescue principle. The small interference RNA capable of reducing SPP1 genetic expression has the sequences shown as SEQ ID NO: 1-2; and the mutant cloning vector, which aims at the target sequences of the RNAi capable of reducing SPP1 genetic expression, is constructed. In the method of the invention, the function change situation is observed by employing RNAi for silencing genes; the rescued mutant vector and RNAi are both used for infecting or transfecting a target cell, so that the endogenesis target gene is silenced, and the protein possessing the same functions with the protein expressed by and endogenesis vector is expressed by the exogenous mutant type vector; the function of the cell is not changed by the above combined processing, or the function is rescued after the mutant type vector is introduced into RNAi and silenced, so that it is proved that the function phenotype is caused by the gene. Therefore, a new model and a method are provided for gene function research.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing mutant cloning vectors based on the principle of RNAi rescue. Background technique [0002] RNAi (RNA interference, RNA interference) is an efficient and specific gene blocking technology, which can silence the target gene by cutting off the mRNA of the target gene. RNAi technology is an emerging gene research tool, which has been widely used Gene function research in various fields. [0003] As the research progresses, it is found that RNAi has relatively common off-target phenomenon. In order to confirm whether the silenced gene is the target gene, people use different RNAi targets to confirm the same gene function results to prove that the target gene has been specifically silenced. There is a possibility that all the designed RNAi targets are off-target, and if all the designed RNAi targets are off-target, it cannot prove whether the target gene has been s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N7/00C12N15/63C12Q1/68
Inventor 曹跃琼朱向莹金杨晟
Owner SHANGHAI GENECHEM
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